September 2016
Volume 57, Issue 12
Open Access
ARVO Annual Meeting Abstract  |   September 2016
Discovery of FetuinA and global signalling/remodelling modules driving myofibroblast differentiation during corneal wound healing in patients
Author Affiliations & Notes
  • Arkasubhra Ghosh
    GROW Research Laboratory, Narayana Nethralaya Foundation, Bangalore, India
    Singapore Eye Research Institute, Singapore, Singapore
  • Krishnatej Nishtala
    GROW Research Laboratory, Narayana Nethralaya Foundation, Bangalore, India
  • Dhananjay Kumar
    GROW Research Laboratory, Narayana Nethralaya Foundation, Bangalore, India
  • rohit shetty
    Cornea and refractive services, Narayana Nethralaya, Bangalore, India
  • Footnotes
    Commercial Relationships   Arkasubhra Ghosh, None; Krishnatej Nishtala, None; Dhananjay Kumar, None; rohit shetty, None
  • Footnotes
    Support  Narayana Nethralaya Foundation
Investigative Ophthalmology & Visual Science September 2016, Vol.57, 1276. doi:
  • Views
  • Share
  • Tools
    • Alerts
      ×
      This feature is available to Subscribers Only
      Sign In or Create an Account ×
    • Get Citation

      Arkasubhra Ghosh, Krishnatej Nishtala, Dhananjay Kumar, rohit shetty; Discovery of FetuinA and global signalling/remodelling modules driving myofibroblast differentiation during corneal wound healing in patients. Invest. Ophthalmol. Vis. Sci. 2016;57(12):1276.

      Download citation file:


      © 2017 Association for Research in Vision and Ophthalmology.

      ×
  • Supplements
Abstract

Purpose : Stromal cell differentiation is an essential process in corneal wound healing. Using quantitative proteomics, we attempt to discover alterations in global protein networks during activation of quiescent keratocytes to active fibroblasts and myofibroblasts.

Methods : Human corneal fibroblasts(HCF) were transformed into keratocytes(KT) by culturing in serum free media for 96h. HCF treated with 1ng/ml TGFβ every 24h for 5 days differentiated to myofibroblasts(MYO). The cell types were identified by specific expression of marker genes Aldh3(KT), Thy-1(HCF) and αSMA(MYO). Cell lysates and patient tear samples were subjected to quantitative proteomic analysis using iTRAQ labelled tandem LC-MS/MS. Tear samples were collected after approval from Institutional Ethics Committee and written, informed consent from corneal wound patients(n=6) and age-matched healthy controls(n=6).

Results : Over 700 proteins were identified with 95% confidence and filtered by >1.5-fold iTRAQ ratio and p<0.05. PANTHER analysis showed cytoskeletal regulation, protein assembly and integrin signalling pathway most significantly altered. A novel TGFβ regulatory protein FetuinA was discovered significantly elevated in MYO compared to KT suggesting a global role in repair phenotype. Actin cytoskeleton regulators COF1, PROF were lower in KT compared to HCF whereas ACTN1, MYL12A, MHC9 were elevated in MYO. Chaperones GRP78, Endoplasmin, HSP90A/B HSP70, etc were also increased in MYO. Differentiation and focal adhesion proteins FN, TLN1, ZYX, FLNA etc were upregulated in HCF and MYO. These proteins were further validated by immunoblotting. Proteomic analysis of tears from patients undergoing corneal wound healing demonstrated elevated FetuinA and cytoskeletal proteins, recapitulating observations from in vitro model.

Conclusions : We report a unique set of differentiation factors for wound healing primarily in cytoskeletal and chaperone signalling modules. We uncover FetuinA as highly elevated in myofibroblasts and corneal wound patient tears indicating its critical role in regulating proper healing by modulating TGF-beta function. The data illustrates distinct interactive protein networks that reveal a specific response to TGFβ (from wound or exogenous addition) mediated differentiation process. These novel proteins can serve as drug targets to treat corneal scarring.

This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.

×
×

This PDF is available to Subscribers Only

Sign in or purchase a subscription to access this content. ×

You must be signed into an individual account to use this feature.

×